Method of preparing enantiomers of indole-2,3-dione-3-oxime derivatives

ABSTRACT

The present invention is directed to a method of preparing enantiomers of indole-2,3-dione-3-oxime derivatives.

TECHNICAL FIELD

The present invention is directed to a method of preparing enantiomersof indole-2,3-dione-3-oxime derivatives.

BACKGROUND ART

Indole-2,3-dione-3-oxime derivatives are useful pharmaceutical products.WO 98/14447 (NeuroSearch) describes indole-2,3-dione-3-oxime derivativesuseful for antagonising the effect of excitatory amino acids. Thesecompounds are prepared by conventional methods of chemical synthesisincluding the step of reacting an 1H-indol-2,3-dion with an aminoderivative. More specifically WO 98/14447 describesindole-2,3-dione-3-oxime derivatives, some of which exist as racemicmixtures.

It is often desirable, and sometimes subject to regulatory demands, toundertake drug development on specific enantiomers rather than racemicdrugs. This rationale is based on the findings that often the desiredcharacteristics of chiral compounds reside with one of its enantiomers,while the other enantiomer might in fact add to a potentialtoxicological effect of the drug.

In order to allow thorough investigation of each enantiomer, processesfor obtaining enantiopure preparations of chiral compounds are ofsignificant importance for drug development.

EP 439779 (Chisso Corp.) describes a process for producing opticallyactive hydroxy lactones using enzymatic transesterification. However, amethod of preparing enantiomers of indole-2,3-dione-3-oxime derivativesis not described.

EP 467132 (Chisso Corp.) describes 4-substituted-2-hydroxybutanoates anda process for producing them. However, a method of preparing enantiomersof indole-2,3-dione-3-oxime derivatives is not described.

SUMMARY OF THE INVENTION

The present invention is directed to a novel method for obtainingenantiopure preparations of chiral indole-2,3-dione-3-oxime derivatives.

Thus, in its first aspect, the invention provides a method of obtainingenantiopure preparations of chiral indole-2,3-dione-3-oxime derivatives(Compounds A or B), which method comprises the subsequent steps of

(i) Reacting an 8-amino-1,2,3,4-tetrahydro-isoquinoline (Compound 9)derivative with chloral hydrate and hydroxylamine hydrochloride to givean N-(1,2,3,4-tetrahydro-isoquinolin-8-yl)-2-hydroxyimino-acetamide(Compound 10) derivative (Step 9);

(ii) Adding sulphuric acid to theN-(1,2,3,4-tetrahydro-isoquinolin-8-yl)-2-hydroxyimino-acetamide(Compound 10) derivative obtained in step (i) (Step 10); and

(iii) Reacting the2,3-dioxo-2,3,6,7,8,9-hexahydro-1H-pyrrolo[3,2-h]isoquinoline (Compound11) derivative obtained in step (ii) with chiral (enantiopure (R) or(S)) α-N,N-diBoc-aminoxy-γ-butyrolactone to obtain the desired chiralend product, i.e. enantiopure (R)— or(S)-2-[2-oxo-1,2,6,7,8,9-hexahydro-pyrrolo[3,2-h]isoquinolin-3-ylideneaminooxy]4-hydroxy-butyricacid) (Compound A or B) (Step 11);

followed by recovery of the desired end product.

In another aspect the invention provides a method of preparing theenantiopure starting material for use according to the method of theinvention which method comprises the subsequent steps of

(i) acetylating a racemic mixture of α-hydroxy-γ-butyrolactone to obtainracemic α-acetoxy-γ-butyrolactone (Step 1);

(ii) subjecting the racemic α-acetoxy-γ-butyrolactone obtained in step(i) to enzymatic de-acetylation to obtain enantiopure (S) or (R)α-acetoxy-γ-butyrolactone (Step 2); and

(iii) subjecting the enantiopure (S) or (R) α-acetoxyy-γ-butyrolactoneobtained in step (ii) to hydrolysis using acidic ion-exchange (Step 3);

followed by recovery of the desired end product.

Other objects of the invention will be apparent to the person skilled inthe art from the following detailed description and examples.

DETAILED DISCLOSURE OF THE INVENTION The Indole-2,3-dione-3-oximeDerivatives

The present invention provides a method of preparing enantiomers ofindole-2,3-dione-3-oxime derivatives. The indole-2,3-dione-3-oximederivatives obtained according to the method of the invention may inparticular be characterised by the general Formula IA or IB

wherein

R represents hydrogen, alkyl or benzyl; and

R⁴ represent hydrogen or alkyl; and

R⁵ represents phenyl, which phenyl may optionally be substituted one ormore times with substituents selected from the group consisting ofhalogen, CF₃, NO₂, CN, NH₂, alkyl, alkoxy and SO₂NR⁵¹R⁵²; wherein

R⁵¹ and R⁵², independently of each another represent hydrogen or alkyl;or

R⁵¹ and R⁵² together with the N-atom to which they are attached form apiperidinyl- or morpholinyl-ring; and

R⁶ and R⁷ together form a benzo-fused ring of the formula—NR⁸—CH₂—CH₂—CH₂—;—CH₂—NR⁸—CH₂—CH₂—;—CH₂—CH₂—NR⁸—CH₂—; or—CH₂—CH₂—CH₂—NR⁸—;

wherein R⁸ represents hydrogen or alkyl.

In a more preferred embodiment the indole-2,3-dione-3-oxime derivativeobtained according to the method of the invention is characterised byFormula IIA or IIB

wherein

R represents hydrogen or alkyl;

R⁵ represents phenyl, which phenyl is optionally substituted withhalogen, CF₃, NO₂, CN or SO₂NR⁵¹R⁵²; wherein

R⁵¹ and R⁵², independently of each another represent hydrogen or alkyl;or

R⁵¹ and R⁵² together with the N-atom to which they are attached form apiperidinyl- or morpholinyl-ring; and

R⁸ represents hydrogen or alkyl.

In an even more preferred embodiment the indole-2,3-dione-3-oximederivative obtained according to the method of the invention ischaracterised by Formula IIIA or IIIB

wherein

R represents hydrogen or alkyl;

R⁵³ represents SO₂NR⁵¹R⁵²; wherein

R⁵¹ and R⁵², independently of each another represent hydrogen or alkyl;or

R⁵¹ and R⁵² together with the N-atom to which they are attached form apiperidinyl- or morpholinyl-ring; and

R⁸ represents hydrogen or alkyl.

In an even more preferred embodiment the indole-2,3-dione-3-oximederivative obtained according to the method of the invention ischaracterised by the general Formula IIIA or IIIB, wherein

R represents hydrogen or C₁₋₃-alkyl;

R⁵³ represents SO₂NR⁵¹R⁵²; wherein

R⁵¹ and R⁵², independently of each another represent hydrogen orC₁₋₃-alkyl; or

R⁵¹ and R⁵² together with the N-atom to which they are attached form apiperidinyl- or morpholinyl-ring; and

R⁸ represents hydrogen or C₁₋₆-alkyl.

In a most preferred embodiment the indole-2,3-dione-3-oxime derivativeobtained according to the method of the invention is Compound IVA or IVB

The Method of Obtaining Enantiopure Preparations of ChiralIndole-2,3-dione-3-oxime Derivatives

The present invention provides a method of preparing enantiomers ofindole-2,3-dione-3-oxime derivatives, in particular theindole-2,3-dione-3-oxime derivatives described above.

In a preferred embodiment the method of the invention comprises thesubsequent steps of

(i) Reacting an 8-amino-1,2,3,4-tetrahydro-isoquinoline (Compound 9)derivative with chloral hydrate and hydroxylamine hydrochloride to givean N-(1,2,3,4-tetrahydro-isoquinolin-8-yl)-2-hydroxyimino-acetamide(Compound 10) derivative (Step 9);

(ii) Adding sulphuric acid to theN-(1,2,3,4-tetrahydro-isoquinolin-8-yl)-2-hydroxyimino-acetamide(Compound 10) derivative obtained in step (i) (Step 10); and

(iii) Reacting the2,3-dioxo-2,3,6,7,8,9-hexahydro-1H-pyrrolo[3,2-h]isoquinoline (Compound11) derivative obtained in step (ii) with chiral (enantiopure (R) or(S)) α-N,N-diBoc-aminoxy-γ-butyrolactone to obtain the desired chiralend product, i.e. enantiopure (R)— or(S)-2-[2-oxo-1,2,6,7,8,9-hexahydro-pyrrolo[3,2-h]isoquinolin-3-ylideneaminooxy]-4-hydroxy-butyricacid) (Compound A or B) (Step 11);

followed by recovery of the desired end product.

In a preferred embodiment the method further comprises the step of

(a) reacting enantiopure (S) or (R) α-hydroxy-γ-butyrolactone withN,N-diBoc-hydroxylamine to give enantiopure (S) or (R)α-N,N-diBoc-aminoxy-γ-butyrolactone (Step 8a);

followed by steps (i) to (iii) as described above.

In another preferred embodiment the method further comprises the step of

(b) subjecting N,N-diBoc-O-benzylhydroxylamine to hydrogenation to giveN,N-diBoc-hydroxylamine (Step 7);

followed by step (a) and steps (i) to (iii) as described above.

In a third preferred embodiment the method further comprises the step of

(c) converting O-benzylhydroxylamine intoN,N-diBoc-O-benzylhydroxylamine using Boc₂O (Step 6);

followed by step (b), step (a), and steps (i) to (iii) as describedabove.

In a fourth preferred embodiment the method further comprises the stepof

(d) reacting enantiopure (S) or (R) α-hydroxy-γ-butyrolactone with tosylchloride to give enantiopure (S) or (R) α-tosyloxy-γ-butyrolactone (Step5);

followed by step (c), step (b), step (a), and steps (i) to (iii) asdescribed above.

In a most preferred embodiment the8-amino-1,2,3,4-tetrahydro-isoquinoline (Compound 9) derivative of step(i) is4-(8-amino-2-methyl-1,2,3,4-tetrahydro-isoquinolin-5-yl)-N,N-dimethyl-benzenesulfonamide(to obtainN-[5-(4-dimethylsulfamoyl-phenyl)-2-methyl-1,2,3,4-tetrahydro-isoquinolin-8-yl]-2-hydroxyimino-acetamide);and

the 2,3-dioxo-2,3,6,7,8,9-hexahydro-1H-pyrrolo[3,2-h]isoquinoline(Compound 11) derivative of step (iii) isN,N-dimethyl-4-(8-methyl-2,3-dioxo-2,3,6,7,8,9-hexahydro-1H-pyrrolo[3,2-h]isoquinolin-5-yl)-benzenesulfonamide;

giving enantiopure (R)- or(S)-2-[5-(4-dimethylsulfamoyl-phenyl)-8-methyl-2-oxo-1,2,6,7,8,9-hexahydro-pyrrolo[3,2-h]isoquinolin-3-ylideneaminooxy]-4-hydroxy-butyricacid as the end product (Compound A or B).

Preparation of the Starting Materials

In another aspect the invention provides a method of producing theenantiopure starting materials for use according to the method describedabove.

The method of the invention for preparing the starting materials ischaracterised by comprising the subsequent steps of

(i) acetylating a racemic mixture of α-hydroxy-γ-butyrolactone to obtainracemic α-acetoxy-γ-butyrolactone (Step 1);

(ii) subjecting the racemic α-acetoxy-γ-butyrolactone obtained in step(i) to enzymatic de-acetylation to obtain enantiopure (S) or (R)α-acetoxy-γ-butyrolactone (Step 2); and

(iii) subjecting the enantiopure (S) or (R) α-acetoxy-γ-butyrolactoneobtained in step (ii) to hydrolysis using acidic ion-exchange (Step 3);

followed by recovery of the desired end product, i.e. the enantiopure(S) or (R) α-hydroxy-γ-butyrolactone.

In a preferred embodiment the method further comprises the step of

(iv) subjecting the enantio-impure remainings of step (iii), i.e. theenantio-impure α-hydroxy-γ-butyrolactone and α-acetoxy-γ-butyrolactone,to racemisation using acid or base;

followed by re-entry of the racemic mixture into step (i).

In a more preferred embodiment the enzymatic de-acetylation of step (ii)is carried out using a lipolytic enzyme. In a most preferred embodimentthe lipolytic enzyme is Lipase PS (available from Amano PharmaceuticalCo.).

EXAMPLE

The invention is further illustrated with reference to the followingexample, which is not intended to be in any way limiting to the scope ofthe invention as claimed.

First the synthetic route to the optical isomers of2-[5-(4dimethylsulfamoyl-phenyl)-8-methyl-2-oxo-1,2,6,7,8,9-hexahydro-pyrrolo[3,2-h]isoquinolin-3-ylideneaminooxy]-4-hydroxy-butyricacid, hereinafter designated Compounds IVA and IVB, is outlined.

Synthesis of the Chiral Building Blocks

Synthesis of the N,N-DiBoc Protected Hydroxylamine Part

Synthesis of the isatin Derivative

Synthesis of Enantiopure (R)-E-1 and (S)-E-1

Next, a more detailed description of each step of the synthesis ispresented.Step 1

A mixture of racemic α-hydroxy-γ-butyrolactone (25 g; 0.25 mol), DMAP(1.5 g), acetylchloride (25.5 ml; 0.37 mol) in CH₂Cl₂ (325 mL) was addedsolid K₂CO₃ (50 g) in portions (slightly exothermic). The reactionmixture was stirred at room temperature overnight, filtered andevaporated to dryness. Column chromatography (Petrol ether80-100:EtOAc=2:1) gave 26.4 g (75%) racemic α-acetoxy-γ-butyrolactone.Step 2

A mixture of racemic a-acetoxyy-butyrolactone (29.8 g; 0.207 mol) andAmano's Lipase PS (800 mg) in THF/water (200 mL; 10/1) was stirred atroom temperature overnight. The reaction mixture was filtered andevaporated to dryness. The crude product was dissolved in CH₂Cl₂ (250mL) and extracted with cold water (5×50 mL). The organic phase was dried(MgSO₄), filtered and evaporated to dryness to yield 12.6 g (43%)enantiopure (S)-α-acetoxy-γ-butyrolactone.

The combined aqueous phases were washed with CH₂Cl₂ (5×50 mL). Theaqueous phase was evaporated to dryness, added CH₂Cl₂, dried (MgSO₄),filtered and evaporated to dryness to give 8.5 g (40%)(R)-α-hydroxy-γ-butyrolactone. Overall yield by moles; 83%. [α]_(D)²⁵=+65.7°.Step 3

A suspension of enantiopure (S)-α-acetoxy-γ-butyrolactone (5 g, 34.7mmol) and Amberlite IRA120 (acid form) in H₂O (50 mL) was heated toreflux and reaction progress monitored by GCMS. After 2 hours, GCMSindicated complete transformation. The reaction mixture was filtered andevaporated to dryness. Vacuum distillation 0.3-0.4 mbar (b.p. 85-92° C.)afforded the enantiopure (S)-α-hydroxy-γ-butyrolactone (2.6 g, 76%yield). [α]_(D) ²⁵=−66.1°.

The crude product may also be isolated from the aqueous solution byextraction with CH₂Cl₂ and the pure product alternatively be isolated bycolumn chromatography. Amberlite IRA-120 may be substituted by H₂SO₄,however with an overall lower yield.

Step 4

Enantio-impure remainings of α-hydroxy-γ-butyrolactone andα-acetoxy-γ-butyrolactone from step 3 may be recycled by racemisationusing acid or base, followed by re-entry into steps 1 to 3.Step 5

A suspension of (R)-α-hydroxy-γ-butyrolactone (3.5 g, 34.3 mmol), tosylchloride (10.0 g, 52.6 mmol), and K₂CO₃ (2.23 g, 16.2 mmol) in dryCH₂Cl₂ (50 mL) was stirred at room temperature overnight. The reactionmixture was filtered, and the filtrate washed with 1M phosphate bufferpH=7 (2×50 mL), 3 M NaCl (aq.) (1×50 mL), dried with Na₂SO₄, filteredand evaporated to dryness to give the crude product. The crude productwas triturated by PE40/60 (50 mL) and stirred for 30 minutes. Theprecipitate formed was isolated by filtration and washed with PE40/60 toremove trace amounts of tosyl chloride, to give 7.7 g (87%) ofenantiopure (R)-α-tosyloxy-γ-butyrolactone. [α]₃₆₅ ²⁵=−9.9°.

The (S)-isomer was prepared similarly. [α]₃₆₅ ²⁵+9.8°.Step 6

A suspension of O-benzylhydroxylamine hydrochloride (30 g, 188 mmol) inacetonitrile (150 mL) was added triethylamine (29 mL). The thicksuspension was stirred for 60 minutes at room temperature and filtered.The precipitate was washed with acetonitrile (100 mL) on the filter. Thecombined filtrates were added to a cooled (0° C.) solution of Boc₂O (45g, 207 mmol) in acetonitrile (150 ml), over 20 minutes. The reactionmixture was stirred at 0° C., and allowed slowly to warm up to roomtemperature. The reaction mixture was left with stirring at roomtemperature overnight, when ¹H-NMR indicated complete conversion ofO-benzylhydroxylamine into N-Boc-O-benzylhydroxylamine.

The reaction mixture was added a solution of Boc₂O (6.7 g, 310 mmol) inacetonitrile (150 mL), followed by a solution of DMAP (2 g, 16.4 mmol)in acetonitrile (30 mL). The reaction mixture was then stirred at 40° C.until TLC (EtOAc:PE60/80=1:5) indicated complete conversion of theintermediate N-Boc-O-benzylhydroxylamine (approx. 2 hours). The reactionmixture was evaporated to dryness and re-dissolved in EtOAc (200 mL).The organic phase was washed with a mixture of 1M phosphate buffer pH=7(100 mL) and NaCl (aq., sat.) (50 mL). The organic phase was dried withNa₂SO₄, filtered and evaporated to dryness to give an oil, whichprecipitated upon trituration by PE40/60 (20 mL). The precipitated wasisolated by filtration to give 56 g (92%) ofN,N-diBoc-O-benzylhydroxylamine as a white solid.Step 7

A suspension of N,N-diBoc-O-benzylhydroxylamine (15 g, 46.4 mmol) and 5%Pd/C (0.9 g) in 96% EtOH was subjected to hydrogenation at atmosphericpressure. When 390 mL H₂ was consumed, hydrogenation was stopped. Thereaction mixture was filtered through celite and evaporated to drynessto give 12.5 g of an orange oil composed of N,N-diBoc-hydroxylamine andN,N-diBoc-imide (86:14).

The oil was dissolved in EtOAc (25 mL) and extracted with 1M NaOH (3×25mL). The combined aqueous phases were immediately poured into a 1Mphosphate buffer pH=7 and extracted with EtOAc (3×75 mL). The combinedorganic fractions were dried with Na₂SO₄, filtered and evaporated todryness. The isolated white solid was air dried to give 8.8 g (81%) ofpure N,N-diBoc-hydroxylamine.Step 8a

A solution of N,N-diBoc-hydroxylamine (8.5 g, 36 mmol) in dry THF (50mL) under N₂ was added Ph₃P (11 g, 42 mmol) and cooled to 0° C. Asolution of (S)-α-hydroxy-γ-butyrolactone (3.6 g, 35 mmol) in dry THF(10 mL) was added, followed by addition of DEAD (6.8 ml, 42 mmol) during30 minutes. The dark reaction mixture was stirred at 0° C. until TLC(EtOAc:PE60/80=1:2, KMnO₄ spray) indicated complete conversion of(S)-α-hydroxy-γ-butyrolactone (approx. 2 hours).

The reaction mixture was evaporated to dryness, dissolved in Et₂O (50mL) and triturated by addition of PE40/60 (100 mL). The reaction mixturewas filtered, and the precipitate thoroughly washed withEt₂O:PE40/60=1:1 (100 mL). The combined organic filtrates wereevaporated to dryness to give 16 g of brown oil. The oil was subjectedto column chromatography. (1000 mL SiO₂, 7 cm in diameter, eluent:EtOAc:PE60/80=1:2). The pure fractions were combined, evaporated todryness and washed with PE40/60 (15 mL) to give 8.5 g (77%) ofenantiopure (R)-α-N,N-diBoc-aminoxy-γ-butyrolactone.

(R)-isomer: [α_(D) ²⁵=+62.5°; [α]₃₆₅ ²⁵=+210°.

The (R)-isomer was prepared similarly: [α_(D) ²⁵=−62.8°; [α]₃₆₅²⁵=−211°.Step 8b

A suspension of 60% NaH (350 mg, 8.8 mmol) in dry CH₂Cl₂ under N₂ at 0°C. was added a solution of BocNHOH in CH₂Cl₂ (5 mL). The reactionmixture was stirred at 0° C. for 30 minutes, where after a solution of(R)-α-tosyloxy-γ-butyrolactone (2 g, 7.8 mmol) in CH₂Cl₂ (5 mL) wasadded. The reaction mixture was allowed to warm up to room temperature,and left with stirring overnight. The reaction mixture was quenched with1M phosphate buffer pH=6.3 (20 mL) and the organic phase separated. Theaqueous phase was extracted with CH₂Cl₂ (2×20 mL), and the combinedorganic fractions extracted with NaCl (aq., sat.) (15 mL). The organicfraction was dried with Na₂SO₄, filtered and evaporated to dryness.Column chromatography (150 g SiO₂, eluent: EtOAc:PE60/80=1:2) gave 1.5 g(89%) of (S)-α-N-Boc-aminoxy-γ-butyrolactone. [α_(D) ²⁵=−42°; [α]₃₆₅²⁵=−127°.

In a similar way the (R)-isomer, containing approximately 10% of(S)-isomer, was prepared: [α]₃₆₅ ²⁵=+70°.Steps 9-10

Purification

Chloral hydrate (71.6 g, 0.434 mol), hydroxylamine hydrochloride (60.3g, 0.868 mol) and Na₂SO₄ (243.3 g, 1.65 mol) were added in sequence to areaction vessel containing H₂O (1.8 L) preheated to 80° C. When a clearsolution was achieved, the aniline(4-(8-amino-2-methyl-1,2,3,4-tetrahydro-isoquinolin-5-yl)-N,N-dimethyl-benzenesulfonamide;99.0 g, 0.217 mol) was added. The reaction was stirred for 45 minuteswhen TLC showed full consumption of the aniline. Celite (39 g) was addedand the reaction mixture was cooled to 40° C. and stirred for 1 hour.The reaction mixture was filtered and the precipitate washed with coldH₂O (500 mL). The isolated solid material (isonitrosoacetanilide oncelite) was dried under a heating lamp to give 132 g material, which wasused without further purification.

The crude isonitrosoacetanilide derivative(N-[5-(4-dimethylsulfamoyl-phenyl)-2-methyl-1,2,3,4-tetrahydro-isoquinolin-8-yl]-2-hydroxyimino-acetamide)obtained above was added over 45 minutes to a solution of H₂SO₄ (350mL), preheated to 75° C. During the addition, the reaction temperatureincreased to 80-85° C. After complete addition, the reaction was stirredfor another 30 minutes at a reaction temperature of 75-85° C., when allthe isonitrosoacetanilide was consumed (TLC, CH₂Cl₂:acetone:MeOH=4:1:1).

The reaction mixture (i.e.N,N-dimethyl-4-(8-methyl-2,3-dioxo-2,3,6,7,8,9-hexahydro-1H-pyrrolo[3,2-h]isoquinolin-5-yl)-benzenesulfonamideand4-(3-hydroxyimino-8-methyl-2-oxo-2,3,6,7,8,9-hexahydro-1H-pyrrolo[3,2-h]isoquinolin-5-yl)-N,N-dimethyl-benzenesulfonamide)was poured onto H₂O (3.5 L), heated to reflux and then filtered warm toremove celite. The celite was washed with boiling water (750 mL), andthe combined aqueous fractions allowed cooling slowly overnight forprecipitation. The precipitate was filtered off, washed with H₂O (2×250mL) and 96% EtOH (2×250 mL) and then air dried to give 69.8 g (67%) ofcrude isatin derivative as the hydrosulfate. The crude product contained11.4% (typically 9-13%) of the oxime.

Crude product (38.1 g, 76.7 mmol) was suspended in H₂O (800 mL) and pHadjusted to 13 using 4 M NaOH (approx. 80 mL) to give a purple solution,which was stirred for 20 minutes. The then yellow-orange solution wasadded THF (300 mL) and pH adjusted to 8.5 using AcOH. This solution waswashed with EtOAc (4×400 mL), then added conc. H₂SO₄ (90 mL) and heatedto 75° C. The aqueous solution was allowed to slowly cool to roomtemperature and then 0° C. The precipitate formed was filtered off, thenwashed with H₂O (2×100 mL) and 96% EtOH (2×100 mL) to give 31.3 g(corresponding to approx. 53% yield from the aniline) of the pure isatinderivative as the hydrosulfate.Step 11

A suspension of the isatin derivative (i.e.N,N-dimethyl-4-(8-methyl-2,3-dioxo-2,3,6,7,8,9-hexahydro-1H-pyrrolo[3,2-h]isoquinolin-5-yl)-benzenesulfonamide)as the hydrosulfate (32.5 g, 65.4 mmol) was suspended in H₂O (650 mL).The reaction mixture was heated to 60° C. and pH adjusted to 1.5 using2M H₂SO₄ (aq.). A solution of (R)- or(S)-α-N,N-diBoc-aminoxy-γ-butyrolactone (23 g, 72.5 mmol) in THF (130mL) was added. The thick reaction mixture was stirred at 60° C. for 9hours, when HPLC indicated complete consumption of the isatinderivative.

The reaction mixture was left overnight at room temperature, warmed to40° C. and pH adjusted to 10 using 1M NaOH (aq.) (approx. 150 mL).Further 1M NaOH (aq.) (approx. 180 mL overall) was added during thereaction to keep pH at 10. After stirring for 4 hours at 40° C., thereaction mixture became a nearly clear solution. HPLC indicated that allproduct, in intermediate lactone form was consumed, and pH was adjustedto 8.5 using 2M H₂SO₄ (aq.).

The reaction mixture was evaporated to dryness to yield 40.3 g solid.The isolated solid was refluxed in a mixture of i-PrOH/H₂O (85/15) (1600mL) and filtered warm (inorganic salts are removed). The filtrate wasallowed to slowly cool to 0° C., and left overnight. The precipitateformed, was isolated by filtration to give 23.9 g yellow to orangecrystals. The crystals (20 g) was dissolved in H₂O (80 mL) and stirredvigorously with EtOAc (250 mL) for 1½ hour (to remove the Z-isomer andremainings of the isatin oxime). The organic phase was discarded, andthe aqueous phase was evaporated to dryness. The solid material isolatedwas suspended in THF (400 mL) and filtered.

The precipitate was dissolved in H₂O (160 mL) and pH adjusted slowly to5.2 using 2M H₂SO₄ (aq.). The mixture was stirred 1½ hour at roomtemperature and filtered. The isolated solid was washed with cold H₂O(2×25 mL) and air dried to give A or B (i.e. (R)— or(S)-2-[5-(4-dimethylsulfamoyl-phenyl)-8-methyl-2-oxo-1,2,6,7,8,9-hexahydro-pyrrolo[3,2-h]isoquinolin-3-ylideneaminooxy]-4-hydroxy-butyricacid) as a yellow amorphous solid, 15.8 g (56%). The reaction occurswith retention of stereochemistry, i.e. no inversion is observed.Step 12

A suspension of isatin derivative 12-1 (i.e.N,N-dimethyl-4-(8-methyl-2,3-dioxo-2,3,6,7,8,9-hexahydro-1H-pyrrolo[3,2-h]isoquinolin-5-yl)-benzenesulfonamide)as the hydrosulfate (3.5 g, 7 mmol) in 96% EtOH (100 ml) was addedhydroxylammonium chloride (1 g, 14 mmol) and then refluxed for 2½ hours.As the starting material was not completely consumed (TLC), furtherhydroxylammonium chloride (0.3 g, 2.1 mmol) was added. The reactionmixture was left at reflux over night.

The reaction mixture was cooled and filtered, and the precipitate washedwith 96% EtOH. The precipitate was suspended in H₂O (100 ml), warmed to75-80° C. and pH adjusted to 8.5 using 4 M NaOH (aq.). The aqueoussolution was cooled to rt. and filtered. The precipitate was washed withH₂O, then suspended in abs. EtOH (100 ml) and brought to reflux. Thesolution was cooled to room temperature and filtered.

The precipitate was washed with abs. EtOH and air dried to give 2.63 g(91%) of 12-2 (i.e.8-methyl-5-(4-(N,N-dimethylsulphamoyl)-phenyl)-6,7,8,9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxime)as the free base. The free base darkens upon standing but may be storedas the hydrosulfate.Step 13

A suspension of 60% NaH (50 mg, 1.25 mmol) in dry DMF (2 ml) under N₂ at0° C. was slowly added a solution of the isatin oxime,8-methyl-5-(4-(N,N-dimethylsulphamoyl)-phenyl)-6,7,8,9-tetrahydro-1H-pyrrolo-[3,2-h]-isoquinoline-2,3-dione-3-oxime(500 mg, 1.25 mmol) in dry DMF (8 ml). The reaction mixture was stirredfor 30 minutes at 0° C., where after a solution of(R)-α-tosyloxy-γ-butyrolactone (340 mg, 1.33 mmol) in dry DMF (2 ml) wasadded. The reaction was left with stirring at room temperatureovernight, when HPLC indicated complete consumption of the isatin oxime.

The reaction mixture was evaporated to dryness and then added H₂O (20ml) and dioxane (5 ml). The reaction mixture was adjusted to pH=10 byuse of 1M NaOH. Further NaOH was added to keep the pH at 10. Thereaction mixture was left with stirring over night at room temperatureand heated to 40-45° C. for 1 hour, when HPLC indicated completeconversion of the intermediate lactone. The reaction mixture wasadjusted to pH=8.5 and evaporated to dryness (extensive foaming).

The isolated solid was refluxed in i-PrOH (15 ml) for 5 minutes, cooledto room temperature and filtered to give 610 mg of solid material. Partof the solid material (550 mg) was refluxed in a mixture of i-PrOH (15ml) and H₂O (0.75 ml) and cooled to 0° C. The precipitate formed wasisolated by filtration to give 310 mg of crude material.

The mother liquor was mainly composed of sodium tosylate. The crudematerial (300 mg) was dissolved in H₂O (10 ml) and pH adjusted to 5.7using 1M HCl, alternatively 5.2 using 2M H₂SO₄ (aq.). An oil separatesand H₂O was removed be decantation. The product precipitates upontrituration by EtOH. The product was isolated by filtration, giving 160mg (28%) of IVA or IVB (i.e. (R)— or(S)-2-[5-(4-dimethylsulfamoyl-phenyl)-8-methyl-2-oxo-1,2,6,7,8,9-hexahydro-pyrrolo[3,2-h]isoquinolin-3-ylideneaminooxy]-4-hydroxy-butyricacid).

The reaction occurs with inversion of stereochemistry, i.e. thestereocenter in α-tosyloxy-γ-butyrolactone is inverted.

1. A method of preparing the chiral (±) isomers ofindole-2,3-dione-3-oxime derivatives (Compounds A or B), which methodcomprises the subsequent steps of (i) Reacting an8-amino-1,2,3,4-tetrahydro-isoquinoline (Compound 9) derivative withchloral hydrate and hydroxylamine hydrochloride to give anN-(1,2,3,4-tetrahydro-isoquinolin-8-yl)-2-hydroxyimino-acetamide(Compound 10) derivative (Step 9); (ii) Adding sulphuric acid to theN-(1,2,3,4-tetrahydro-isoquinolin-8-yl)-2-hydroxyimino-acetamide(Compound 10) derivative obtained in step (i) (Step 10); and (iii)Reacting the2,3-dioxo-2,3,6,7,8,9-hexahydro-1H-pyrrolo[3,2-h]isoquinoline (Compound11) derivative obtained in step (ii) with chiral (enantiopure (R) or(S)) α-N,N-diBoc-aminoxy-γ-butyrolactone to obtain the desired chiralend product, i.e. enantiopure (R)- or(S)-2-[2-oxo-1,2,6,7,8,9-hexahydro-pyrrolo[3,2-h]isoquinolin-3-ylideneaminooxy]-4-hydroxy-butyricacid) (Compound A or B) (Step 11); followed by recovery of the desiredend product.
 2. The method of claim 1, which method further comprisesthe step of (a) reacting enantiopure (S) or (R)α-hydroxy-γ-butyrolactone with N,N-diBoc-hydroxylamine to giveenantiopure (S) or (R) α-N,N-diBoc-aminoxy-γ-butyrolactone (Step 8a);followed by steps (i) to (iii) of claim
 1. 3. The method of claim 2,which method further comprises the step of (b) subjectingN,N-diBoc-O-benzylhydroxylamine to hydrogenation to giveN,N-diBoc-hydroxylamine (Step 7); followed by step (a) of claim 2, andsteps (i) to (iii) of claim
 1. 4. The method of claim 3, which methodfurther comprises the step of (c) converting O-benzylhydroxylamine intoN,N-diBoc-O-benzylhydroxylamine using Boc₂O (Step 6); followed by step(b) of claim 3, step (a) of claim 2, and steps (i) to (iii) of claim 1.5. The method of claim 1, which method further comprises the step of (d)reacting enantiopure (S) or (R) α-hydroxy-γ-butyrolactone with tosylchloride to give enantiopure (S) or (R) α-tosyloxy-γ-butyrolactone (Step5); followed by step (c) of claim 4, step (b) of claim 3, step (a) ofclaim 2, and steps (i) to (iii) of claim
 1. 6. The method of claim 1,wherein the 8-amino-1,2,3,4-tetrahydro-isoquinoline (Compound 9)derivative of step (i) is4-(8-amino-2-methyl-1,2,3,4-tetrahydro-isoquinolin-5-yl)-N,N-dimethyl-benzenesulfonamide(to obtainN-[5-(4-dimethylsulfamoyl-phenyl)-2-methyl-1,2,3,4-tetrahydro-isoquinolin-8-yl]-2-hydroxyimino-acetamide);and the 2,3-dioxo-2,3,6,7,8,9-hexahydro-1H-pyrrolo[3,2-h]isoquinoline(Compound 11) derivative of step (iii) isN,N-dimethyl-4-(8-methyl-2,3-dioxo-2,3,6,7,8,9-hexahydro-1H-pyrrolo[3,2-h]isoquinolin-5-yl)-benzenesulfonamide;giving enantiopure (R)— or(S)-2-[5-(4-dimethylsulfamoyl-phenyl)-8-methyl-2-oxo-1,2,6,7,8,9-hexahydro-pyrrolo[3,2-h]isoquinolin-3-ylideneaminooxy]-4-hydroxy-butyricacid as the end product (Compound A or B).
 7. A method of preparing astarting material for use according to the method of claim 1, whichmethod comprises the subsequent steps of (i) acetylating a racemicmixture of α-hydroxy-γ-butyrolactone to obtain racemicα-acetoxy-γ-butyrolactone (Step 1); (ii) subjecting the racemiccc-acetoxy-y-butyrolactone obtained in step (i) to enzymaticde-acetylation to obtain enantiopure (S) or (R)α-acetoxy-γ-butyrolactone (Step 2); and (iii) subjecting the enantiopure(S) or (R) α-acetoxy-γ-butyrolactone obtained in step (ii) to hydrolysisusing acidic ion-exchange (Step 3); followed by recovery of the desiredend product.
 8. The method of claim 7, which method further comprisesthe step of (iv) subjecting the enantio-impure remainings of step (iii),i.e. the enantio-impure α-hydroxy-γ-butyrolactone andα-acetoxy-γ-butyrolactone, to racemisation using acid or base; followedby re-entry of the racemic mixture into step (i).
 9. The method of claim7, wherein the enzymatic de-acetylation of step (ii) is carried outusing a lipolytic enzyme.
 10. Enantiopure(R)-2-[5-(4-dimethylsulfamoyl-phenyl)-8-methyl-2-oxo-1,2,6,7,8,9-hexahydro-pyrrolo[3,2-h]isoquinolin-3-ylideneaminooxy]-4-hydroxy-butyricacid.
 11. Enantiopure(S)-2-[5-(4-dimethylsulfamoyl-phenyl)-8-methyl-2-oxo-1,2,6,7,8,9-hexahydro-pyrrolo[3,2-h]isoquinolin-3-ylideneaminooxy]-4-hydroxy-butyricacid.